Yina H. Huang

Themis controls thymocyte selection through regulation of T cell antigen receptor–mediated signaling

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre–T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

Production of Ins(1,3,4,5)P4 mediated by the kinase Itpkb inhibits store-operated calcium channels and regulates B cell selection and activation.

Antigen receptor–mediated production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in lymphocytes triggers the release of Ca2+ from intracellular stores; this release of Ca2+ results in the opening of store-operated Ca2+ channels in the plasma membrane. Here we report that mice lacking Ins(1,4,5)P3 3-kinase B (Itpkb), which converts Ins(1,4,5)P3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), had impaired B lymphocyte development and defective immunoglobulin G3 antibody responses to a T lymphocyte–independent antigen. Itpkb-deficient B lymphocytes had the phenotypic and functional features of tolerant B lymphocytes and showed enhanced activity of store-operated Ca2+ channels after B lymphocyte receptor stimulation, which was reversed by the provision of exogenous Ins(1,3,4,5)P4. Our data identify Itpkb and its product Ins(1,3,4,5)P4 as inhibitors of store-operated Ca2+ channels and crucial regulators of B cell selection and activation.

Notch signaling in lymphocyte development.

Signaling through Notch has been implicated in many cell-fate decisions during lymphocyte development. Recent studies have provided new clues--and raised new controversies--regarding the exact role that Notch signaling plays in the commitment of cells to the T-cell lineage. Progress has also been made in deducing the transcriptional program induced by Notch and the mechanism of oncogenic transformation by Notch in lymphocytes.

The Wnt/Planar Cell Polarity Pathway Component Vangl2 Induces Synapse Formation through Direct Control of N-Cadherin

Although regulators of the Wnt/planar cell polarity (PCP) pathway are widely expressed in vertebrate nervous systems, their roles at synapses are unknown. Here, we show that Vangl2 is a postsynaptic factor crucial for synaptogenesis and that it coprecipitates with N-cadherin and PSD-95 from synapse-rich brain extracts. Vangl2 directly binds N-cadherin and enhances its internalization in a Rab5-dependent manner. This physical and functional interaction is suppressed by β-catenin, which binds the same intracellular region of N-cadherin as Vangl2. In hippocampal neurons expressing reduced Vangl2 levels, dendritic spine formation as well as synaptic marker clustering is significantly impaired. Furthermore, Prickle2, another postsynaptic PCP component, inhibits the N-cadherin-Vangl2 interaction and is required for normal spine formation. These results demonstrate direct control of classic cadherin by PCP factors; this control may play a central role in the precise formation and maturation of cell-cell adhesions at the synapse.

Dynamic Repositioning of CD4 and CD8 Genes during T Cell Development

Although stable repression of CD4 and CD8 genes is a central feature of T cell lineage commitment, we lack detailed information about the timing and mechanism of this repression. Stable gene repression has been linked to the position of genes within the nucleus. Therefore, information about the nuclear position of CD4 and CD8 genes during T cell development could provide insights into both the mechanism of regulation of CD4 and CD8 genes, and the process of lineage commitment. Here, we report that lineage-specific repression of CD4 and CD8 genes is associated with the repositioning of alleles close to heterochromatin. We also provide evidence that the relocalization of CD4 and CD8 genes to heterochromatin can occur as an early response to positive selection signals. We discuss our results in terms of our current knowledge of CD4 and CD8 gene regulation and CD4 versus CD8 lineage commitment.

Resident memory T cells in the skin mediate durable immunity to melanoma

Resident memory CD8 T cells maintained in vitiligo-affected skin mediate long-lived protection against melanoma. Resident memory to cancer Melanoma patients with vitiligo are more likely to have a positive outcome, but the mechanism behind this association has remained unclear. Now, Malik et al. report that skin-resident memory T (TRM) cells specific to melanoma antigens are maintained in vitiligo-affected skin. These cells persist and function independently of the lymphoid compartment, suggesting that the vitiligo lesions provide a niche for the TRM cells. The TRM cells provide durable memory to the tumor, even in pigmented skin. These data suggest that skin TRM cells are critical for maintaining antitumor immunity. Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen–specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein–1) or LAG-3 (lymphocyte activation gene–3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.

Melanoma Induces, and Adenosine Suppresses, CXCR3-Cognate Chemokine Production and T-cell Infiltration of Lungs Bearing Metastatic-like Disease

Clancy-Thompson and colleagues show that lung metastatic-like melanoma induces a transient production of CXCR3-cognate chemokines and IFN required for antigen-specific T-cell infiltration into the tumor site, which in part is temporally limited by adenosine signaling and reversible by the adenosine receptor antagonist aminophylline. Despite immunogenicity, melanoma-specific vaccines have demonstrated minimal clinical efficacy in patients with established disease but enhanced survival when administered in the adjuvant setting. Therefore, we hypothesized that organs bearing metastatic-like melanoma may differentially produce T-cell chemotactic proteins over the course of tumor development. Using an established model of metastatic-like melanoma in lungs, we assessed the production of specific cytokines and chemokines over a time course of tumor growth, and we correlated chemokine production with chemokine receptor–specific T-cell infiltration. We observed that the interferon (IFN)-inducible CXCR3-cognate chemokines (CXCL9 and CXCL10) were significantly increased in lungs bearing minimal metastatic lesions, but chemokine production was at or below basal levels in lungs with substantial disease. Chemokine production was correlated with infiltration of the organ compartment by adoptively transferred CD8+ tumor antigen-specific T cells in a CXCR3- and host IFNγ-dependent manner. Adenosine signaling in the tumor microenvironment (TME) suppressed chemokine production and T-cell infiltration in the advanced metastatic lesions, and this suppression could be partially reversed by administration of the adenosine receptor antagonist aminophylline. Collectively, our data demonstrate that CXCR3-cognate ligand expression is required for efficient T-cell access of tumor-infiltrated lungs, and these ligands are expressed in a temporally restricted pattern that is governed, in part, by adenosine. Therefore, pharmacologic modulation of adenosine activity in the TME could impart therapeutic efficacy to immunogenic but clinically ineffective vaccine platforms. Cancer Immunol Res; 3(8); 956–67. ©2015 AACR.

New therapeutic targets in immune disorders: ItpkB, Orai1 and UNC93B

Background: Sequencing of the murine and human genomes has enabled large-scale functional genomics approaches to target identification. This holds the promise of drastically accelerating target discovery. Moreover, by providing an initial validation coincident with target identification, cell based cDNA or small interfering RNA (siRNA) screens and in particular genome-wide in vivo approaches, including forward or reverse genetics and analyses of natural gene polymorphisms, can move the relatively late step of target validation to the beginning of the process, reducing the risk of pursuing targets with little in vivo relevance. Objective: We critically discuss the value of combining functional genomics with traditional approaches for accelerating target identification and validation. Methods: We evaluate the potentials of inositol (1,4,5)trisphosphate 3-kinase B (ItpkB), Orai1 and UNC93B, three particularly interesting proteins that were recently identified through functional genomics, as targets in immune disorders. Results/conclusion: Combining functional genomics with traditional approaches can accelerate target discovery and validation, but requires a follow-up platform that integrates and analyzes all relevant data for assessment of the clinical potential of the growing number of novel targets.

Inositol tetrakisphosphate limits NK cell effector functions by controlling PI3K signaling

Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell-based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP(3) generation by PI3K and generation of diacylglycerol and IP(3) by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP(3) metabolite inositol (1,3,4,5)tetrakisphosphate (IP(4)) in NK cells. IP(4) promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP(4) limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP(3) effector-kinase Akt. This identifies IP(4) as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP(4) is a broadly important signaling paradigm.

Calmodulin and PI(3,4,5)P3 cooperatively bind to the Itk pleckstrin homology domain to promote efficient calcium signaling and IL-17A production

Calcium and lipids stimulate the kinase Itk through the same domain to promote proinflammatory T cell signaling. Two Signals Are Better Than One Lipid binding to pleckstrin homology (PH) domains enables spatial and temporal control of protein activity. T cell activation is a highly localized event and involves calcium signals and PH domain–containing kinases, such as Itk. Wang et al. report that calcium and lipids converged at Itk, creating a positive feedback loop necessary for full T cell activation. Nuclear magnetic resonance (NMR) analysis revealed that the PH domain of Itk interacted with the calcium-binding protein calmodulin and with PI(3,4,5)P3 and that the two enhanced each other’s binding to the PH domain. Both interactions were required for maximal production of the proinflammatory cytokine IL-17A. Furthermore, calmodulin binding occurred with several other PH domains, suggesting that responding to calcium signaling may be a common function of this domain. Precise regulation of the kinetics and magnitude of Ca2+ signaling enables this signal to mediate diverse responses, such as cell migration, differentiation, vesicular trafficking, and cell death. We showed that the Ca2+-binding protein calmodulin (CaM) acted in a positive feedback loop to potentiate Ca2+ signaling downstream of the Tec kinase family member Itk. Using NMR (nuclear magnetic resonance), we mapped CaM binding to two loops adjacent to the lipid-binding pocket within the Itk pleckstrin homology (PH) domain. The Itk PH domain bound synergistically to Ca2+/CaM and the lipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], such that binding to Ca2+/CaM enhanced the binding to PI(3,4,5)P3 and vice versa. Disruption of CaM binding attenuated Itk recruitment to the membrane and diminished release of Ca2+ from the endoplasmic reticulum. Moreover, disruption of this feedback loop abrogated Itk-dependent production of the proinflammatory cytokine IL-17A (interleukin-17A) by CD4+ T cells. Additionally, we found that CaM associated with PH domains from other proteins, indicating that CaM may regulate other PH domain–containing proteins.